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The temporal lobe of the human brain contains the entorhinal cortex (EC). This region of the brain is a highly interconnected integrative hub for sensory and spatial information; it also has a key role in episodic memory formation and is the main source of cortical hippocampal inputs1–4. The human EC continues to develop during childhood5, but neurogenesis and neuronal migration to the EC are widely considered to be complete by birth. Here we show that the human temporal lobe contains many young neurons migrating into the postnatal EC and adjacent regions, with a large tangential stream persisting until the age of around one year and radial dispersal continuing until around two to three years of age. By contrast, we found no equivalent postnatal migration in rhesus macaques (Macaca mulatta). Immunostaining and single-nucleus RNA sequencing of ganglionic eminence germinal zones, the EC stream and the postnatal EC revealed that most migrating cells in the EC stream are derived from the caudal ganglionic eminence and become LAMP5+RELN+ inhibitory interneurons. These late-arriving interneurons could continue to shape the processing of sensory and spatial information well into postnatal life, when children are actively interacting with their environment. The EC is one of the first regions of the brain to be affected in Alzheimer's disease, and previous work has linked cognitive decline to the loss of LAMP5+RELN+ cells6,7. Our investigation reveals that many of these cells arrive in the EC through a major postnatal migratory stream in early childhood. A stream of young neurons migrating into the entorhinal cortex (EC) continues postnatally in humans, but not in macaques; these young neurons, which belong to a unique class of local circuit cells, continue to be recruited in the EC during infancy and early childhood. [ABSTRACT FROM AUTHOR]

Self-organizing three-dimensional cellular models derived from human pluripotent stem cells or primary tissue have great potential to provide insights into how the human nervous system develops, what makes it unique and how disorders of the nervous system arise, progress and could be treated. Here, to facilitate progress and improve communication with the scientific community and the public, we clarify and provide a basic framework for the nomenclature of human multicellular models of nervous system development and disease, including organoids, assembloids and transplants.The nomenclature for human multicellular models of nervous system development and disease, including organoids, assembloids and transplants, is discussed and a consensus framework is presented. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a flavivirus transmitted via mosquitoes and sex to cause congenital neurodevelopmental defects, including microcephaly. Inherited forms of microcephaly (MCPH) are associated with disrupted centrosome organization. Similarly, we found that ZIKV infection disrupted centrosome organization. ZIKV infection disrupted the organization of centrosomal proteins including CEP63, a MCPH‐associated protein. The ZIKV nonstructural protein NS3 bound CEP63, and expression of NS3 was sufficient to alter centrosome architecture and CEP63 localization. Loss of CEP63 suppressed ZIKV‐induced centrosome disorganization, indicating that ZIKV requires CEP63 to disrupt centrosome organization. ZIKV infection or CEP63 loss decreased the centrosomal localization and stability of TANK‐binding kinase 1 (TBK1), a regulator of the innate immune response. ZIKV infection also increased the centrosomal accumulation of the CEP63 interactor DTX4, a ubiquitin ligase that degrades TBK1. Therefore, we propose that ZIKV disrupts CEP63 function to increase centrosomal DTX4 localization and destabilization of TBK1, thereby tempering the innate immune response. Synopsis: Brazilian Zika virus (ZIKV) infection disrupts centrosome organization and destabilizes TBK1, thereby dampening innate immune response activation. ZIKV protein NS3 localizes to the centrosome and redistributes the microcephaly‐associated protein CEP63 from the centrosome.Acentrosomal CEP63 recruits the ubiquitin ligase DTX4 leading to the degradation of TBK1, a regulator of innate immunity.TBK1 loss prevents the activation of the innate immune response in ZIKV‐infected cells. [ABSTRACT FROM AUTHOR]

Organoids are 3D cell culture systems derived from human pluripotent stem cells that contain tissue resident cell types and reflect features of early tissue organization. Neural organoids are a particularly innovative scientific advance given the lack of accessibility of developing human brain tissue and intractability of neurological diseases. Neural organoids have become an invaluable approach to model features of human brain development that are not well reflected in animal models. Organoids also hold promise for the study of atypical cellular, molecular, and genetic features that underscore neurological diseases. Additionally, organoids may provide a platform for testing therapeutics in human cells and are a potential source for cell replacement approaches to brain injury or disease. Despite the promising features of organoids, their broad utility is tempered by a variety of limitations yet to be overcome, including lack of high-fidelity cell types, limited maturation, atypical physiology, and lack of arealization, features that may limit their reliability for certain applications. [ABSTRACT FROM AUTHOR]

Radial glial cells, the stem cells of the cerebral cortex, extend a long basal fiber that ends in basal endfeet. A new study in PLOS Biology found that non-muscle myosins control basal endfoot integrity to regulate interneuron organization. Radial glial cells, the stem cells of the cerebral cortex, extend a long basal fiber that ends in basal endfeet. This Primer explores the implications of a new PLOS Biology study revealing that non-muscle myosins control basal endfoot integrity to regulate interneuron organization. [ABSTRACT FROM AUTHOR]

Studies of the spatiotemporal, transcriptomic, and morphological diversity of radial glia (RG) have spurred our current models of human corticogenesis. In the developing cortex, neural intermediate progenitor cells (nIPCs) are a neuron-producing transit-amplifying cell type born in the germinal zones of the cortex from RG. The potential diversity of the nIPC population, that produces a significant portion of excitatory cortical neurons, is understudied, particularly in the developing human brain. Here we explore the spatiotemporal, transcriptomic, and morphological variation that exists within the human nIPC population and provide a resource for future studies. We observe that the spatial distribution of nIPCs in the cortex changes abruptly around gestational week (GW) 19/20, marking a distinct shift in cellular distribution and organization during late neurogenesis. We also identify five transcriptomic subtypes, one of which appears at this spatiotemporal transition. Finally, we observe a diversity of nIPC morphologies that do not correlate with specific transcriptomic subtypes. These results provide an analysis of the spatiotemporal, transcriptional, and morphological diversity of nIPCs in developing brain tissue and provide an atlas of nIPC subtypes in the developing human cortex that can benchmark in vitro models of human development such as cerebral organoids and help inform future studies of how nIPCs contribute to cortical neurogenesis. [ABSTRACT FROM AUTHOR]

Background: Studies of individuals with autism spectrum disorder (ASD) have revealed a strong multigenic basis with the identification of hundreds of ASD susceptibility genes. ASD is characterized by social deficits and a range of other phenotypes, implicating complex genetics and involvement of a variety of brain regions. However, how mutations and mis-expression of select gene sets are associated with the behavioral components of ASD remains unknown. We reasoned that for genes to be associated with ASD core behaviors they must be: (1) expressed in brain regions relevant to ASD social behaviors and (2) expressed during the ASD susceptible window of brain development. Methods: Focusing on the amygdala, a brain region whose dysfunction has been highly implicated in the social component of ASD, we mined publicly available gene expression databases to identify ASD-susceptibility genes expressed during human and mouse amygdala development. We found that a large cohort of known ASD susceptibility genes is expressed in the developing human and mouse amygdala. We further performed analysis of single-nucleus RNA-seq (snRNA-seq) data from microdissected amygdala tissue from five ASD and five control human postmortem brains ranging in age from 4 to 20 years to elucidate cell type specificity of amygdala-expressed genes and their dysregulation in ASD. Results: Our analyses revealed that of the high-ranking ASD susceptibility genes, 80 are expressed in both human and mouse amygdala during fetal to early postnatal stages of development. Our human snRNA-seq analyses revealed cohorts of genes with altered expression in the ASD amygdala postnatally, especially within excitatory neurons, with dysregulated expression of seven genes predicted from our datamining pipeline. Limitations: We were limited by the ages for which we were able to obtain human tissue; therefore, the results from our datamining pipeline approach will require validation, to the extent possible, in human tissue from earlier developmental stages. Conclusions: Our pipeline narrows down the number of amygdala-expressed genes possibly involved in the social pathophysiology of ASD. Our human single-nucleus gene expression analyses revealed that ASD is characterized by changes in gene expression in specific cell types in the early postnatal amygdala. [ABSTRACT FROM AUTHOR]

Accurate RNA quantification at the single-cell level is critical for understanding the dynamics of gene expression and regulation across space and time. Single molecule FISH (smFISH), such as RNAscope, provides spatial and quantitative measurements of individual transcripts, therefore, can be used to explore differential gene expression among a heterogeneous cell population if combined with cell identify information. However, such analysis is not straightforward, and existing image analysis pipelines cannot integrate both RNA transcripts and cellular staining information to automatically output cell type-specific gene expression. We developed an efficient and customizable analysis method, Single-Molecule Automatic RNA Transcription Quantification (SMART-Q), to enable the analysis of gene transcripts in a cell type-specific manner. SMART-Q efficiently infers cell identity information from multiplexed immuno-staining and quantifies cell type-specific transcripts using a 3D Gaussian fitting algorithm. Furthermore, we have optimized SMART-Q for user experiences, such as flexible parameters specification, batch data outputs, and visualization of analysis results. SMART-Q meets the demands for efficient quantification of single-molecule RNA and can be widely used for cell type-specific RNA transcript analysis. [ABSTRACT FROM AUTHOR]

Cortical organoids are self-organizing three-dimensional cultures that model features of the developing human cerebral cortex1,2. However, the fidelity of organoid models remains unclear3–5. Here we analyse the transcriptomes of individual primary human cortical cells from different developmental periods and cortical areas. We find that cortical development is characterized by progenitor maturation trajectories, the emergence of diverse cell subtypes and areal specification of newborn neurons. By contrast, organoids contain broad cell classes, but do not recapitulate distinct cellular subtype identities and appropriate progenitor maturation. Although the molecular signatures of cortical areas emerge in organoid neurons, they are not spatially segregated. Organoids also ectopically activate cellular stress pathways, which impairs cell-type specification. However, organoid stress and subtype defects are alleviated by transplantation into the mouse cortex. Together, these datasets and analytical tools provide a framework for evaluating and improving the accuracy of cortical organoids as models of human brain development. Single-cell RNA sequencing clarifies the development and specification of neurons in the human cortex and shows that cell stress impairs this process in cortical organoids. [ABSTRACT FROM AUTHOR]

Neuroserpin is a serine‐protease inhibitor mainly expressed in the CNS and involved in the inhibition of the proteolytic cascade. Animal models confirmed its neuroprotective role in perinatal hypoxia‐ischaemia and adult stroke. Although neuroserpin may be a potential therapeutic target in the treatment of the aforementioned conditions, there is still no information in the literature on its distribution during human brain development. The present study provides a detailed description of the changing spatiotemporal patterns of neuroserpin focusing on physiological human brain development. Five stages were distinguished within our examined age range which spanned from the 7th gestational week until adulthood. In particular, subplate and deep cortical plate neurons were identified as the main sources of neuroserpin production between the 25th gestational week and the first postnatal month. Our immunohistochemical findings were substantiated by single cell RNA sequencing data showing specific neuronal and glial cell types expressing neuroserpin. The characterization of neuroserpin expression during physiological human brain development is essential for forthcoming studies which will explore its involvement in pathological conditions, such as perinatal hypoxia‐ischaemia and adult stroke in human. [ABSTRACT FROM AUTHOR]

Chronic neurodegeneration and acute injuries lead to neuron losses via diverse processes. We compared retinal ganglion cell (RGC) responses between chronic glaucomatous conditions and the acute injury model. Among major RGC subclasses, αRGCs and intrinsically photosensitive RGCs (ipRGCs) preferentially survive glaucomatous conditions, similar to findings in the retina subject to axotomy. Focusing on an αRGC intrinsic factor, Osteopontin (secreted phosphoprotein 1 [Spp1]), we found an ectopic neuronal expression of Osteopontin (Spp1) in other RGCs subject to glaucomatous conditions. This contrasted with the Spp1 downregulation subject to axotomy. αRGC-specific Spp1 elimination led to significant αRGC loss, diminishing their resiliency. Spp1 overexpression led to robust neuroprotection of susceptible RGC subclasses under glaucomatous conditions. In contrast, Spp1 overexpression did not significantly protect RGCs subject to axotomy. Additionally, SPP1 marked adult human RGC subsets with large somata and SPP1 expression in the aqueous humor correlated with glaucoma severity. Our study reveals Spp1's role in mediating neuronal resiliency in glaucoma. [Display omitted] • αRGCs and ipRGCs are resilient retinal neuron types in glaucomatous and axotomy conditions • Elevated and ectopic Spp1 expression among RGCs occurs in the glaucoma model, not axotomy • Neuronal Spp1 drives RGC neuroprotection in glaucomatous conditions but not in axotomy • SPP1 protein levels in aqueous humor correlate with glaucoma progression in patients Zhao et al. reported that retinal neuron subpopulations are resilient with ectopic Spp1 expression, subject to an experimental glaucomatous model with ocular hypertension. Spp1 overexpression renders other susceptible retinal neurons with neuroprotection subject to glaucomatous conditions. Elevated SPP1 levels in aqueous humor may be biomarkers for human glaucoma progression. [ABSTRACT FROM AUTHOR]

Background: High throughput methods for profiling the transcriptomes of single cells have recently emerged as transformative approaches for large-scale population surveys of cellular diversity in heterogeneous primary tissues. However, the efficient generation of such atlases will depend on sufficient sampling of diverse cell types while remaining cost-effective to enable a comprehensive examination of organs, developmental stages, and individuals. Results: To examine the relationship between sampled cell numbers and transcriptional heterogeneity in the context of unbiased cell type classification, we explored the population structure of a publicly available 1.3 million cell dataset from E18.5 mouse brain and validated our findings in published data from adult mice. We propose a computational framework for inferring the saturation point of cluster discovery in a single-cell mRNA-seq experiment, centered around cluster preservation in downsampled datasets. In addition, we introduce a "complexity index," which characterizes the heterogeneity of cells in a given dataset. Using Cajal-Retzius cells as an example of a limited complexity dataset, we explored whether the detected biological distinctions relate to technical clustering. Surprisingly, we found that clustering distinctions carrying biologically interpretable meaning are achieved with far fewer cells than the originally sampled, though technical saturation of rare populations such as Cajal-Retzius cells is not achieved. We additionally validated these findings with a recently published atlas of cell types across mouse organs and again find using subsampling that a much smaller number of cells recapitulates the cluster distinctions of the complete dataset. Conclusions: Together, these findings suggest that most of the biologically interpretable cell types from the 1.3 million cell database can be recapitulated by analyzing 50,000 randomly selected cells, indicating that instead of profiling few individuals at high "cellular coverage," cell atlas studies may instead benefit from profiling more individuals, or many time points at lower cellular coverage and then further enriching for populations of interest. This strategy is ideal for scenarios where cost and time are limited, though extremely rare populations of interest (< 1%) may be identifiable only with much higher cell numbers. [ABSTRACT FROM AUTHOR]

New neurons continue to be generated in the subgranular zone of the dentate gyrus of the adult mammalian hippocampus. This process has been linked to learning and memory, stress and exercise, and is thought to be altered in neurological disease. In humans, some studies have suggested that hundreds of new neurons are added to the adult dentate gyrus every day, whereas other studies find many fewer putative new neurons. Despite these discrepancies, it is generally believed that the adult human hippocampus continues to generate new neurons. Here we show that a defined population of progenitor cells does not coalesce in the subgranular zone during human fetal or postnatal development. We also find that the number of proliferating progenitors and young neurons in the dentate gyrus declines sharply during the first year of life and only a few isolated young neurons are observed by 7 and 13 years of age. In adult patients with epilepsy and healthy adults (18-77 years; n = 17 post-mortem samples from controls; n = 12 surgical resection samples from patients with epilepsy), young neurons were not detected in the dentate gyrus. In the monkey (Macaca mulatta) hippocampus, proliferation of neurons in the subgranular zone was found in early postnatal life, but this diminished during juvenile development as neurogenesis decreased. We conclude that recruitment of young neurons to the primate hippocampus decreases rapidly during the first years of life, and that neurogenesis in the dentate gyrus does not continue, or is extremely rare, in adult humans. The early decline in hippocampal neurogenesis raises questions about how the function of the dentate gyrus differs between humans and other species in which adult hippocampal neurogenesis is preserved. [ABSTRACT FROM AUTHOR]

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme ( GBM) is the most common and aggressive type of primary brain tumor. Epidermal growth factor ( EGF) and platelet-derived growth factor ( PDGF) receptors are frequently amplified and/or possess gain-of-function mutations in GBM. However, clinical trials of tyrosine-kinase inhibitors have shown disappointing efficacy, in part due to intra-tumor heterogeneity. To assess the effect of clonal heterogeneity on gene expression, we derived an approach to map single-cell expression profiles to sequentially acquired mutations identified from exome sequencing. Using 288 single cells, we constructed high-resolution phylogenies of EGF-driven and PDGF-driven GBMs, modeling transcriptional kinetics during tumor evolution. Descending the phylogenetic tree of a PDGF-driven tumor corresponded to a progressive induction of an oligodendrocyte progenitor-like cell type, expressing pro-angiogenic factors. In contrast, phylogenetic analysis of an EGFR-amplified tumor showed an up-regulation of pro-invasive genes. An in-frame deletion in a specific dimerization domain of PDGF receptor correlates with an up-regulation of growth pathways in a proneural GBM and enhances proliferation when ectopically expressed in glioma cell lines. In-frame deletions in this domain are frequent in public GBM data. [ABSTRACT FROM AUTHOR]

During mammalian neocortical development, newborn excitatory and inhibitory neurons must migrate over long distances to reach their final positions within the cortical plate. In the lissencephalic rodent brain, pyramidal neurons are born in the ventricular and subventricular zones of the pallium and migrate along radial glia fibers to reach the appropriate cortical layer. Although much less is known about neuronal migration in species with a gyrencephalic cortex, retroviral studies in the ferret and primate suggest that, unlike the rodent, pyramidal neurons do not follow strict radial pathways and instead can disperse horizontally. However, the means by which pyramidal neurons laterally disperse remain unknown. In this study, we identified a viral labeling technique for visualizing neuronal migration in the ferret, a gyrencephalic carnivore, and found that migration was predominantly radial at early postnatal ages. In contrast, neurons displayed more tortuous migration routes with a decreased frequency of cortical plate-directed migration at later stages of neurogenesis concomitant with the start of brain folding. This was accompanied by neurons migrating sequentially along several different radial glial fibers, suggesting a mode by which pyramidal neurons may laterally disperse in a folded cortex. These findings provide insight into the migratory behavior of neurons in gyrencephalic species and provide a framework for using nonrodent model systems for studying neuronal migration disorders. [ABSTRACT FROM AUTHOR]

Evolutionary expansion of the human neocortex underlies many of our unique mental abilities. This expansion has been attributed to the increased proliferative potential of radial glia (RG; neural stem cells) and their subventricular dispersion from the periventricular niche during neocortical development. Such adaptations may have evolved through gene expression changes in RG. However, whether or how RG gene expression varies between humans and other species is unknown. Here we show that the transcriptional profiles of human and mouse neocortical RG are broadly conserved during neurogenesis, yet diverge for specific signalling pathways. By analysing differential gene co-expression relationships between the species, we demonstrate that the growth factor PDGFD is specifically expressed by RG in human, but not mouse, corticogenesis. We also show that the expression domain of PDGFRβ, the cognate receptor for PDGFD, is evolutionarily divergent, with high expression in the germinal region of dorsal human neocortex but not in the mouse. Pharmacological inhibition of PDGFD-PDGFRβ signalling in slice culture prevents normal cell cycle progression of neocortical RG in human, but not mouse. Conversely, injection of recombinant PDGFD or ectopic expression of constitutively active PDGFRβ in developing mouse neocortex increases the proportion of RG and their subventricular dispersion. These findings highlight the requirement of PDGFD-PDGFRβ signalling for human neocortical development and suggest that local production of growth factors by RG supports the expanded germinal region and progenitor heterogeneity of species with large brains. [ABSTRACT FROM AUTHOR]

Non-neurogenic cell types, such as cortical astroglia and fibroblasts, can be directly converted into neurons by the overexpression of defined transcription factors. Normally, the cellular phenotype of such differentiated cells is remarkably stable and resists direct cell transdifferentiation. Here we show that the Ink4a/Arf (also known as Cdkn2a) locus is a developmental barrier to direct neuronal transdifferentiation induced by transcription factor overexpression. With serial passage in vitro, wild-type postnatal cortical astroglia become progressively resistant to Dlx2-induced neuronal transdifferentiation. In contrast, the neurogenic competence of Ink4a/Arfdeficient astroglia is both greatly increased and does not diminish through serial cell culture passage. Electrophysiological analysis further demonstrates the neuronal identity of cells induced from Ink4a/Arf-null astroglia, and short hairpin RNA-mediated acute knockdown of p16Ink4a and p19Arf p16Ink4a and p19Arf indicates that these gene products function postnatally as a barrier to cellular transdifferentiation. Finally, we found that mouse fibroblasts deficient for Ink4a/Arf also exhibit greatly enhanced transcription factorinduced neuronal induction. These data indicate that Ink4a/Arf is a potent barrier to direct neuronal transdifferentiation and further suggest that this locus functions normally in the progressive developmental restriction of postnatal astrocytes. [ABSTRACT FROM AUTHOR]

Evolutionary expansion of the human neocortex is partially attributed to a relative abundance of neural stem cells in the fetal brain called outer radial glia (oRG). oRG cells display a characteristic division mode, mitotic somal translocation (MST), in which the soma rapidly translocates toward the cortical plate immediately prior to cytokinesis. MST may be essential for progenitor zone expansion, but the mechanism of MST is unknown, hindering exploration of its function in development and disease. Here, we show that MST requires activation of the Rho effector ROCK and nonmuscle myosin II, but not intact microtubules, centrosomal translocation into the leading process, or calcium influx. MST is independent of mitosis and distinct from interkinetic nuclear migration and saltatory migration. Our findings suggest that disrupted MST may underlie neurodevelopmental diseases affecting the Rho-ROCK-myosin pathway and provide a foundation for future exploration of the role of MST in neocortical development, evolution, and disease. [ABSTRACT FROM AUTHOR]

Ring chromosomes are structural aberrations commonly associated with birth defects, mental disabilities and growth retardation. Rings form after fusion of the long and short arms of a chromosome, and are sometimes associated with large terminal deletions. Owing to the severity of these large aberrations that can affect multiple contiguous genes, no possible therapeutic strategies for ring chromosome disorders have been proposed. During cell division, ring chromosomes can exhibit unstable behaviour leading to continuous production of aneuploid progeny with low viability and high cellular death rate. The overall consequences of this chromosomal instability have been largely unexplored in experimental model systems. Here we generated human induced pluripotent stem cells (iPSCs) from patient fibroblasts containing ring chromosomes with large deletions and found that reprogrammed cells lost the abnormal chromosome and duplicated the wild-type homologue through the compensatory uniparental disomy (UPD) mechanism. The karyotypically normal iPSCs with isodisomy for the corrected chromosome outgrew co-existing aneuploid populations, enabling rapid and efficient isolation of patient-derived iPSCs devoid of the original chromosomal aberration. Our results suggest a fundamentally different function for cellular reprogramming as a means of 'chromosome therapy' to reverse combined loss-of-function across many genes in cells with large-scale aberrations involving ring structures. In addition, our work provides an experimentally tractable human cellular system for studying mechanisms of chromosomal number control, which is of critical relevance to human development and disease. [ABSTRACT FROM AUTHOR]

The dramatic increase in neocortical size and folding during mammalian brain evolution has been attributed to the elaboration of the subventricular zone (SVZ) and the associated increase in neural progenitors. However, recent studies have shown that SVZ size and the abundance of resident progenitors do not directly predict cortical topography, suggesting that complex behaviors of the progenitors themselves may contribute to the overall size and shape of the adult cortex. Using time-lapse imaging, we examined the dynamic behaviors of SVZ progenitors in the ferret, a gyrencephalic carnivore, focusing our analysis on outer radial glial cells (oRGs). We identified a substantial population of oRGs by marker expression and their unique mode of division, termed mitotic somal translocation (MST). Ferret oRGs exhibited diverse behaviors in terms of division location, cleavage angle, and MST distance, as well as fiber orientation and dynamics. We then examined the human fetal cortex and found that a subset of human oRGs displayed similar characteristics, suggesting that diversity in oRG behavior may be a general feature. Similar to the human, ferret oRGs underwent multiple rounds of self-renewing divisions but were more likely to undergo symmetric divisions that expanded the oRG population, as opposed to producing intermediate progenitor cells (IPCs). Differences in oRG behaviors, including proliferative potential and daughter cell fates, may con- tribute to variations in cortical structure between mammalian species. [ABSTRACT FROM AUTHOR]

GABAergic cortical interneurons underlie the complexity of neural circuits and are particularly numerous and diverse in humans. In rodents, cortical interneurons originate in the subpallial ganglionic eminences, but their developmental origins in humans are controversial. We characterized the developing human ganglionic eminences and found that the subventricular zone (SVZ) expanded massively during the early second trimester, becoming densely populated with neural stem cells and intermediate progenitor cells. In contrast with the cortex, most stem cells in the ganglionic eminence SVZ did not maintain radial fibers or orientation. The medial ganglionic eminence exhibited unique patterns of progenitor cell organization and clustering, and markers revealed that the caudal ganglionic eminence generated a greater proportion of cortical interneurons in humans than in rodents. On the basis of labeling of newborn neurons in slice culture and mapping of proliferating interneuron progenitors, we conclude that the vast majority of human cortical interneurons are produced in the ganglionic eminences, including an enormous contribution from non-epithelial SVZ stem cells. [ABSTRACT FROM AUTHOR]

The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6+ cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6+ cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b), Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP+ cells, while enhancer 1056 is active in Olig2+ cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments. [ABSTRACT FROM AUTHOR]

A hallmark of mammalian brain evolution is cortical expansion, which reflects an increase in the number of cortical neurons established by the progenitor cell subtypes present and the number of their neurogenic divisions. Recent studies have revealed a new class of radial glia-like (oRG) progenitor cells in the human brain, which reside in the outer subventricular zone. Expansion of the subventricular zone and appearance of oRG cells may have been essential evolutionary steps leading from lissencephalic to gyrencephalic neocortex. Here we show that oRG-like progenitor cells are present in the mouse embryonic neocortex. They arise from asymmetric divisions of radial glia and undergo self-renewing asymmetric divisions to generate neurons. Moreover, mouse oRG cells undergo mitotic somal translocation whereby centrosome movement into the basal process during interphase precedes nuclear translocation. Our finding of oRG cells in the developing rodent brain fills a gap in our understanding of neocortical expansion. [ABSTRACT FROM AUTHOR]

Radial glial progenitor cells exhibit bidirectional cell cycle-dependent nuclear oscillations. The purpose and underlying mechanism of this unusual 'interkinetic nuclear migration' are poorly understood. We investigated the basis for this behavior by live imaging of nuclei, centrosomes and microtubules in embryonic rat brain slices, coupled with the use of RNA interference (RNAi) and the myosin inhibitor blebbistatin. We found that nuclei migrated independent of centrosomes and unidirectionally away from or toward the ventricular surface along microtubules, which were uniformly oriented from the ventricular surface to the pial surface of the brain. RNAi directed against cytoplasmic dynein specifically inhibited nuclear movement toward the apical surface. An RNAi screen of kinesin genes identified Kif1a, a member of the kinesin-3 family, as the motor for basally directed nuclear movement. These observations provide direct evidence that kinesins are involved in nuclear migration and neurogenesis and suggest that a cell cycle-dependent switch between distinct microtubule motors drives interkinetic nuclear migration. [ABSTRACT FROM AUTHOR]

Oligodendrocytes and astrocytes are macroglial cells of the vertebrate central nervous system. These cells have diverse roles in the maintenance of neurological function. In the embryo, the genetic mechanisms that underlie the specification of macroglial precursors in vivo appear strikingly similar to those that regulate the development of the diverse neuron types. The switch from producing neuronal to glial subtype-specific precursors can be modelled as an interplay between region-restricted components and temporal regulators that determine neurogenic or gliogenic phases of development, contributing to glial diversity. Gaining insight into the developmental genetics of macroglia has great potential to improve our understanding of a variety of neurological disorders in humans. [ABSTRACT FROM AUTHOR]

The adult cerebral cortex is composed of excitatory and inhibitory neurons that arise from progenitor cells in disparate proliferative regions in the developing brain and follow different migratory paths. Excitatory pyramidal neurons originate near the ventricle and migrate radially to their position in the cortical plate along radial glial fibers. On the other hand, inhibitory interneurons arise in the ventral telencephalon and migrate tangentially to enter the developing cortex before migrating radially to reach their correct laminar position. Gap junction adhesion has been shown to play an important mechanistic role in the radial migration of excitatory neurons. We asked whether a similar mechanism governs the tangential or radial migration of inhibitory interneurons. Using short hairpin RNA knockdown of Connexin 43 (Cx43) and Cx26 together with rescue experiments, we found that gap junctions are dispensable for the tangential migration of interneurons, but that Cx43 plays a role in the switch from tangential to radial migration that allows interneurons to enter the cortical plate and find their correct laminar position. Moreover this action is dependent on the adhesive properties and the C terminus of Cx43 but not the Cx43 channel. Thus, the radial phase of interneuron migration resembles that of excitatory neuron migration in terms of dependence on Cx43 adhesion. Furthermore, gap junctions between migrating interneurons and radial processes were observed by electron microscopy. These findings provide mechanistic and structural support for a gap junction-mediated interaction between migrating interneurons and radial glia during the switch from tangential to radial migration. [ABSTRACT FROM AUTHOR]

Neurons in the developing rodent cortex are generated from radial glial cells that function as neural stem cells. These epithelial cells line the cerebral ventricles and generate intermediate progenitor cells that migrate into the subventricular zone (SVZ) and proliferate to increase neuronal number. The developing human SVZ has a massively expanded outer region (OSVZ) thought to contribute to cortical size and complexity. However, OSVZ progenitor cell types and their contribution to neurogenesis are not well understood. Here we show that large numbers of radial glia-like cells and intermediate progenitor cells populate the human OSVZ. We find that OSVZ radial glia-like cells have a long basal process but, surprisingly, are non-epithelial as they lack contact with the ventricular surface. Using real-time imaging and clonal analysis, we demonstrate that these cells can undergo proliferative divisions and self-renewing asymmetric divisions to generate neuronal progenitor cells that can proliferate further. We also show that inhibition of Notch signalling in OSVZ progenitor cells induces their neuronal differentiation. The establishment of non-ventricular radial glia-like cells may have been a critical evolutionary advance underlying increased cortical size and complexity in the human brain. [ABSTRACT FROM AUTHOR]

Of the many signals in the developing nervous system, GABA (γ-aminobutyric acid) has been shown to be one of the earliest neurotransmitters present. Unlike in the adult, where this transmitter acts synaptically to inhibit neurons, during development, GABA can depolarize progenitor cells and their progeny due to their high intracellular chloride concentration. This early form of GABA signalling may provide the main excitatory drive for the immature cortical network and play a central role in regulating cortical development. Many features of GABA signalling are conserved in different species and are recapitulated during neurogenesis in the adult brain, demonstrating the importance of this versatile molecule in driving cortical formation. Here, we present recent evidence supporting the multiple functions of GABA during embryonic development and adult neurogenesis, from regulating progenitor proliferation to influencing the migration and maturation of newborn neurons. [ABSTRACT FROM AUTHOR]

Summary: About 30% of patient with epilepsy do not respond to available antiepileptic drugs. In addition to seizure suppression, novel approaches are needed to prevent or alleviate epileptogenic process after various types of brain injuries. The use of cell transplants as factories to produce endogeneous anticonvulsants or as bricks to repair abnormal ictogenic and epileptogenic neuronal circuits has generated hope that cell-based therapies could become a novel therapeutic category in the treatment arsenal of epilepsy. Herein we summarize the current status and future perspectives of cell-based therapies in the treatment of epilepsy. [Copyright &y& Elsevier]

The development of a balance between excitatory and inhibitory synapses is a critical process in the generation and maturation of functional circuits. Accumulating evidence suggests that neuronal activity plays an important role in achieving such a balance in the developing cortex, but the mechanism that regulates this process is unknown. During development, GABA, the primary inhibitory neurotransmitter in adults, excites neurons as a result of high expression of the Na+-K+-2Cl- cotransporter (NKCC1). Using NKCC1 RNA interference knockdown in vivo, we show that GABA-induced depolarization is necessary for proper excitatory synapse formation and dendritic development of newborn cortical neurons. Blocking NKCC1 with the diuretic bumetanide during development leads to similar persistent changes in cortical circuitry in the adult. Interestingly, expression of a voltage-independent NMDA receptor rescues the failure of NKCC1 knockdown neurons to develop excitatory AMPA transmission, indicating that GABA depolarization cooperates with NMDA receptor activation to regulate excitatory synapse formation. Our study identifies an essential role for GABA in the synaptic integration of newborn cortical neurons and suggests an activity-dependent mechanism for achieving the balance between excitation and inhibition in the developing cortex [ABSTRACT FROM AUTHOR]

Radial glia, the neuronal stem cells of the embryonic cerebral cortex, reside deep within the developing brain and extend radial fibres to the pial surface, along which embryonic neurons migrate to reach the cortical plate. Here we show that the gap junction subunits connexin 26 (Cx26) and connexin 43 (Cx43) are expressed at the contact points between radial fibres and migrating neurons, and acute downregulation of Cx26 or Cx43 impairs the migration of neurons to the cortical plate. Unexpectedly, gap junctions do not mediate neuronal migration by acting in the classical manner to provide an aqueous channel for cell–cell communication. Instead, gap junctions provide dynamic adhesive contacts that interact with the internal cytoskeleton to enable leading process stabilization along radial fibres as well as the subsequent translocation of the nucleus. These results indicate that gap junction adhesions are necessary for glial-guided neuronal migration, raising the possibility that the adhesive properties of gap junctions may have an important role in other physiological processes and diseases associated with gap junction function. [ABSTRACT FROM AUTHOR]

Two distinct populations of cerebral cortical progenitor cells that generate neurons during embryogenesis have been identified: radial glial cells and intermediate progenitor cells. Despite advances in our understanding of progenitor cell populations, we know relatively little about factors that regulate their proliferative behaviour. 17-β-Estradiol (E2) is present in the adult and developing mammalian brain, and plays an important role in central nervous system processes such as neuronal differentiation, survival and plasticity. E2 also stimulates neurogenesis in the adult dentate gyrus. We examined the role of E2 during embryonic cortical neurogenesis through immunohistochemistry, in situ hybridization, functional enzyme assay, organotypic culture and in utero administration of estradiol-blocking agents in mice. We show that aromatase, the E2 synthesizing enzyme, is present in the embryonic neocortex, that estrogen receptor-α is present in progenitor cells during cortical neurogenesis, that in vitro E2 administration rapidly promotes proliferation, and that in utero blockade of estrogen receptors decreases proliferation of embryonic cortical progenitor cells. Furthermore, the E2 inhibitor α-fetoprotein is expressed at high levels by radial glial cells but at lower levels by intermediate progenitor cells, suggesting that E2 differentially influences the proliferation of these cortical progenitor cell types. These findings demonstrate a new functional role for E2 as a proliferative agent during critical stages of cerebral cortex development. [ABSTRACT FROM AUTHOR]

Our knowledge of the proliferation, migration, and differentiation of neurons has changed dramatically over the last 10 years. Whereas traditionally it was thought that glial and neuronal cells were separate cell lines with different lineages, we now know that this is not true. Radial glia are a type of neural stem cell that generate excitatory pyramidal neurons directly through asymmetric cell division in the ventricular zone (VZ) of the telencephalon and indirectly through the symmetric division of daughter intermediate precursor cells that divide in the subventricular zone (SVZ). Moreover, pyramidal neurons, once thought to migrate only along radial guide fibers to the developing layers of the cortex, have been shown to proceed through four distinct stages of migration during which they change shape, direction, and speed. Gamma-aminobutyric acid (GABAergic) inhibitory interneurons, on the other hand, are generated not in the cortex, but in the medial ganglionic eminence and migrate tangentially to their final cortical destinations. Evidence suggests that GABA activation may play a role in coordinating the generation and migration of both pyramidal and interneuron populations. At the end of neurogenesis, radial glial cells translocate to the cortex and transform into astrocytes. Although they do not actively divide in the adult brain, astrocytes may retain the potential to generate new neurons. These new findings have increased our understanding of the mechanisms underlying certain developmental disorders and, in doing so, reveal potentially useful modes of therapeutic intervention. [ABSTRACT FROM AUTHOR]

Precise patterns of cell division and migration are crucial to transform the neuroepithelium of the embryonic forebrain into the adult cerebral cortex. Using time-lapse imaging of clonal cells in rat cortex over several generations, we show here that neurons are generated in two proliferative zones by distinct patterns of division. Neurons arise directly from radial glial cells in the ventricular zone (VZ) and indirectly from intermediate progenitor cells in the subventricular zone (SVZ). Furthermore, newborn neurons do not migrate directly to the cortex; instead, most exhibit four distinct phases of migration, including a phase of retrograde movement toward the ventricle before migration to the cortical plate. These findings provide a comprehensive and new view of the dynamics of cortical neurogenesis and migration. [ABSTRACT FROM AUTHOR]

In the mature brain, GABA (γ-aminobutyric acid) functions primarily as an inhibitory neurotransmitter. But it can also act as a trophic factor during nervous system development to influence events such as proliferation, migration, differentiation, synapse maturation and cell death. GABA mediates these processes by the activation of traditional ionotropic and metabotropic receptors, and probably by both synaptic and non-synaptic mechanisms. However, the functional properties of GABA receptor signalling in the immature brain are significantly different from, and in some ways opposite to, those found in the adult brain. The unique features of the early-appearing GABA signalling systems might help to explain how GABA acts as a developmental signal. [ABSTRACT FROM AUTHOR]

Summary: Purpose: Cortical dysplasia (CD) is associated with epilepsy in both the pediatric and adult populations. The mechanism underlying seizures with cortical malformations is still poorly understood. To study the physiology of dysplastic cortex, we developed an experimental model of CD. Methods: Pregnant rats were given intraperitoneal injections of carmustine (1-3-bis-chloroethyl-nitrosourea; BCNU) on embryonic day 15 (E15). Cortical histology was examined in the resulting pups at P0, P28, and P60. In addition, evoked and spontaneous field potential recordings were obtained in cortical slices from adult control and BCNU-exposed rats. Finally, we used whole-cell recordings to compare physiologic properties of pyramidal neurons and γ-aminobutyric acid (GABA) responses in control and BCNU-treated animals. Results: Features characteristic of CD were found in the offspring, including laminar disorganization, cytomegalic neurons, and neuronal heterotopias. Dysplastic cortex also contained abnormal clusters of Cajal–Retzius (CR) cells and disruption of radial glial fibers, as demonstrated with immunohistochemistry. Under conditions of partial GABAA -receptor blockade with 10 μM bicuculline methiodide (BMI), slices of dysplastic cortex demonstrated a significant increase in the number of spontaneous and evoked epileptiform discharges. Individual pyramidal neurons in dysplastic cortex were less sensitive to application of GABA compared with controls. Conclusions: BCNU exposure in utero produces histologic alterations suggestive of CD in rat offspring. Dysplastic cortex from this model demonstrates features of hyperexcitability and decreased neuronal sensitivity to GABA. Such physiologic alterations may underlie the increased epileptogenicity of dysplastic cortex. [ABSTRACT FROM AUTHOR]

Demonstrates that neurons derived from radial glial cells establish radial units in the brain's neocortex. Demonstration that clones consist of mitotic radial glia and postmitotic neurons; Neurons migration along clonally-related radial glia; Support for the concept that a lineage relationship between neurons and proliferative radial glia may underlie the neocortex's radial organization.